MicroRNA-181a-5p Suppresses Cell Proliferation By Targeting Egr1 And Inhibiting Egr1/TGF-?/Smad Pathway In Hepatocellular Carcinoma

Backgroud and ObjectiveHepatocellular carcinoma(HCC)is the leading cause of cancer mortality worldwide.Early growth response factor 1(Egrl)plays a crucial role in cancer progression.However,its precise role and underlying mechanism in HCC has not been clear.MicroRNAs(miRNAs)have been gradually proved to play important roles in HCC.However,it is not clear that which miRNA can affect HCC progression by binding to Egr1 3'UTR.We performed the present study including in vivo and in vitro experiments to clarify the role of Egrl in cell proliferation of HCC and the underlying molecular mechanism and which miRNA can affect on the progression of HCC through suppressing Egrl expression.We hope that this study will improve the better understanding of HCC pathogenesis and the development of effective therapies for HCC.MethodsWe firstly analyzed the immumohistochemical staining of Egrl and TGF-?1 in HCC from the human protein atlas(http://www.proteinatlas.org).Twenty pairs of fresh HCC and adjacent non-tumorous liver samples were obtained from the patients who received surgery at the Second Affiliated Hospital of Jinan University from 2015 to 2016.The expressions of Egr1 and TGF-?1 were dectected through qRT-PCR and Western blot analysis.Human HCC cell lines HepG2 and Hep3B were used to conduct in vitro experiments.Cells were treated with pENTER-Egr1 plasmid or siEgr1 respectively.TGF-?1 expression was measured by qRT-PCR and Western blot analysis.The protein levels of p-Smad2,Smad2,p-Smad3,Smad3 were detected by western blot.CCK-8 assays were conducted to determine the viability of transfected cells.Public available algorithms was performed to identify the miRNAs that may target Egr1 3'UTR.The expressions of miRNAs were measured through qRT-PCR analysis in 20 paired HCC specimens.Spearman's correlation coefficient was used to calculate the correlations between miRNA expression and Egr1 level.MiR-181a-5p was determined in this study.Dual-luciferase activity assay was conducted to confirm the relationship between miR-181 a-5p and Egr1 in HCC.HepG2 cells were transfected with miR-181a-5p mimics and Hep3B cells with inhibitor respectively.The expressions of Egr1 and TGF-?1 were detected by qRT-PCR and Western blot analysis.The viability of transfected cells were measured through CCK-8 assays.SiEgr1 together with miR-181a-5p inhibitor were transfected into HepG2 and Hep3B cells.The levels of Egr1 and TGF-?1 were determined by qRT-PCR and Western blot analysis.The viability of transfected cells were detected through CCK-8 assays.ResultsThe human protein atlas(http://www.proteinatlas.org)showed that Egr1 and TGF-?1 had the positive strong expression in HCCs and negative weak expression in normal liver tissues.Furthermore,mRNA and protein levels of Egr1 and TGF-?1 were dramatically increased in 20 paired HCC tissues.It was shown that up-regulated Egrl significantly increased the expression of TGF-?1 and caused a significant increase in the phosphorylation levels of Smad2 and Smad3.The cell growth curves demonstrated that overexpression of Egrl enhanced cell proliferation in HepG2 and Hep3B cells.In contrast,silenced Egrl expression resulted in decreased expressions of TGF-(31 and phosphor-Smad2(p-Smad2)and phosphor-Smad3(p-Smad3).After treating with siEgrl,the proliferative ability of HepG2 and Hep3B cells were significantly reduced.We used public available algorithms(PicTar,http://pictar.mdc-belin.de/;TargetScan,www.targetscan.org/;and miRanda,www.microrma.org)to predict that miR-181a-5p and other 8 miRNAs were identified to have potential target sites in 3'UTR of Egrl mRNA.Among these,miR-181a-5p was proved to down-regulate in 20 paired HCC tissues.In the meanwhile,a significant inverse correlation between miR-181a-5p expression and Egrl mRNA expression in 20 paired HCC tissues was confirmed with Pearson correlation analysis(R=0.435,P = 0.0016).Dual-luciferase activity assay demonstrated definitely that miR-181 a-5p can target Egrl 3'UTR.It was shown that elevated expression of miR-181a-5p significantly down-regulated the mRNA and protein levels of Egrl and TGF-?1 and suppressed the viability of HCC cells.Conversely,decreased expression of miR-181a-5p revealed noticeably increased levels of Egrl and TGF-?1 and enhanced viability of HCC cells.The decreased expression of Egrl and TGF-?1 were restored when Egrl-silenced cells were treated with miR-181a-5p inhibitor.In the meanwhile,the suppressed cell viability induced by siEgrl was rescued by miR-181a-5p inhibitor in HepG2 and Hep3B cells.Conclusions1.The elevated expression of Egrl promoted the cell proliaferation in HCC.2.Egrl enhanced tumor cells proliferation by activating TGF-?1/Smad signaling pathway in HCC.3.MiR-181a-5p suppressed Egrl expression through targeting Egrl 3'UTR directly.4.MiR-181a-5p/Egrl/TGF-?1/Smad signaling pathway played an important role in HCC progression.